PROJECT SUMMARY Prokaryotes use an adaptive immune system called CRISPR-Cas to defend against phages and other mobile genetic elements. These systems acquire short pieces of DNA from their invaders as a genetic record of prior infections. They use these to produce CRISPR RNAs that are assembled with one or more Cas proteins into RNA-guided interference complexes that find and cleave a complementary target in foreign DNA or RNA. While most types of CRISPR-Cas systems target only one type of nucleic acid, Type III CRISPR systems have been reported to target both DNA and RNA. The identity of the true target nucleic acid of Type III systems thus remains unknown. A bacterial Type III-A system was reported to target transcriptionally active loci in vivo, leading to the hypothesis that the transcription bubble, at which DNA and RNA are both present, is the physiological target. Target recognition by the interference complex thus may involve simultaneous recognition of both DNA and RNA. In order to test this model, we propose to 1) trap a Type III-A (Csm) complex bound to a transcriptionally active target and use cryo-electron microscopy to determine the structure of the complex and 2) use protein-nucleic acid binding and nucleic acid cleavage assays to determine if RNA binding recruits the Type III-A (Csm) complex to the transcription bubble. This would lay the groundwork for repurposing these systems to detect transcriptionally active loci, and regulate gene expression in heterologous organisms.